Replication stress promotes cell elimination by extrusion.

Cell extrusion is a mechanism of cell elimination that is used by organisms as diverse as sponges, nematodes, insects and mammals1-3. During extrusion, a cell detaches from a layer of surrounding cells while maintaining the continuity of that layer4. Vertebrate epithelial tissues primarily eliminate cells by extrusion, and the dysregulation of cell extrusion has been linked to epithelial diseases, including cancer1,5. The mechanisms that drive cell extrusion remain incompletely understood. Here, to analyse cell extrusion by Caenorhabditis elegans embryos3, we conducted a genome-wide RNA interference screen, identified multiple cell-cycle genes with S-phase-specific function, and performed live-imaging experiments to establish how those genes control extrusion. Extruding cells experience replication stress during S phase and activate a replication-stress response via homologues of ATR and CHK1. Preventing S-phase entry, inhibiting the replication-stress response, or allowing completion of the cell cycle blocked cell extrusion. Hydroxyurea-induced replication stress6,7 triggered ATR-CHK1- and p53-dependent cell extrusion from a mammalian epithelial monolayer. We conclude that cell extrusion induced by replication stress is conserved among animals and propose that this extrusion process is a primordial mechanism of cell elimination with a tumour-suppressive function in mammals.

Lipid transporter Spns2 promotes microglia pro-inflammatory activation in response to amyloid-beta peptide.

Accumulating evidence indicates that neuroinflammation contributes to the pathogenesis and exacerbation of neurodegenerative disorders, such as Alzheimer's disease (AD). Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid that regulates many pathophysiological processes including inflammation. We present evidence here that the spinster homolog 2 (Spns2), a S1P transporter, promotes microglia pro-inflammatory activation in vitro and in vivo. Spns2 knockout (Spns2KO) in primary cultured microglia resulted in significantly reduced levels of pro-inflammatory cytokines induced by lipopolysaccharide (LPS) and amyloid-beta peptide 1-42 oligomers (Aß42) when compared with littermate controls. Fingolimod (FTY720), a S1P receptor 1 (S1PR1) functional antagonist and FDA approved drug for relapsing-remitting multiple sclerosis, partially blunted Aß42-induced pro-inflammatory cytokine generation, suggesting that Spns2 promotes microglia pro-inflammatory activation through S1P-signaling. Spns2KO significantly reduced Aß42-induced nuclear factor kappa B (NFκB) activity. S1P increased, while FTY720 dampened, Aß42-induced NFκB activity, suggesting that Spns2 activates microglia inflammation through, at least partially, NFκB pathway. Spns2KO mouse brains showed significantly reduced Aß42-induced microglia activation/accumulation and reduced levels of pro-inflammatory cytokines when compared with age-matched controls. More interestingly, Spns2KO ameliorated Aß42-induced working memory deficit detected by Y-Maze. In summary, these results suggest that Spns2 promotes pro-inflammatory polarization of microglia and may play a crucial role in AD pathogenesis.

Novel function of ceramide for regulation of mitochondrial ATP release in astrocytes.

We reported that amyloid ß peptide (Aß42) activated neutral SMase 2 (nSMase2), thereby increasing the concentration of the sphingolipid ceramide in astrocytes. Here, we show that Aß42 induced mitochondrial fragmentation in wild-type astrocytes, but not in nSMase2-deficient cells or astrocytes treated with fumonisin B1 (FB1), an inhibitor of ceramide synthases. Unexpectedly, ceramide depletion was concurrent with rapid movements of mitochondria, indicating an unknown function of ceramide for mitochondria. Using immunocytochemistry and super-resolution microscopy, we detected ceramide-enriched and mitochondria-associated membranes (CEMAMs) that were codistributed with microtubules. Interaction of ceramide with tubulin was confirmed by cross-linking to N-[9-(3-pent-4-ynyl-3-H-diazirine-3-yl)-nonanoyl]-D-erythro-sphingosine (pacFACer), a bifunctional ceramide analog, and binding of tubulin to ceramide-linked agarose beads. Ceramide-associated tubulin (CAT) translocated from the perinuclear region to peripheral CEMAMs and mitochondria, which was prevented in nSMase2-deficient or FB1-treated astrocytes. Proximity ligation and coimmunoprecipitation assays showed that ceramide depletion reduced association of tubulin with voltage-dependent anion channel 1 (VDAC1), an interaction known to block mitochondrial ADP/ATP transport. Ceramide-depleted astrocytes contained higher levels of ATP, suggesting that ceramide-induced CAT formation leads to VDAC1 closure, thereby reducing mitochondrial ATP release, and potentially motility and resistance to Aß42 Our data also indicate that inhibiting ceramide generation may protect mitochondria in Alzheimer's disease.

Increased liver tumor formation in neutral sphingomyelinase-2-deficient mice.

Sphingolipids are key signaling lipids in cancer. Genome-wide studies have identified neutral SMase-2 (nSMase2), an enzyme generating ceramide from SM, as a potential repressor for hepatocellular carcinoma. However, little is known about the sphingolipids regulated by nSMase2 and their roles in liver tumor development. We discovered growth of spontaneous liver tumors in 27.3% (9 of 33) of aged male nSMase2-deficient (fro/fro) mice. Lipidomics analysis showed a marked increase of SM in the tumor. Unexpectedly, tumor tissues presented with more than a 7-fold increase of C16-ceramide, concurrent with upregulation of ceramide synthase 5. The fro/fro liver tumor, but not adjacent tissue, exhibited substantial accumulation of lipid droplets, suggesting that nSMase2 deficiency is associated with tumor growth and increased neutral lipid generation in the tumor. Tumor tissue expressed significantly increased levels of CD133 and EpCAM mRNA, two markers of liver cancer stem-like cells (CSCs) and higher levels of phosphorylated signal transducer and activator of transcription 3, an essential regulator of stemness. CD133(+) cells showed strong labeling for SM and ceramide. In conclusion, these results suggest that SMase-2 deficiency plays a role in the survival or proliferation of CSCs, leading to spontaneous tumors, which is associated with tumor-specific effects on lipid homeostasis.

Secretory expression and enzymatic characterization of recombinant Agarivorans albus ß-agarase in Escherichia coli.

Agarase catalyzes the hydrolysis of agar, which is primarily used as a medium for microbiology, various food additives, and new biomass materials. In this study, we described the expression of the synthetic gene encoding ß-agarase from Agarivorans albus (Aaß-agarase) in Escherichia coli. The synthetic ß-agarase gene was designed based on the biased codons of E. coli to optimize its expression and extracellular secretion in an active, soluble form. The synthesized agarase gene, including its signal sequence, was cloned into the pET-26 expression vector, and the pET-Aaß-agarase plasmid was introduced into E. coli BL21-Star (DE3) cells. The E. coli transformants were cultured for high-yield secretion of recombinant Aaß-agarase in Luria-Bertani broth containing 0.6 mM isopropyl ß-D-1-thiogalactopyranoside for 9 h at 37°C. The expressed recombinant Aaß-agarase was purified by ammonium sulfate precipitation and diethylaminoethyl-sepharose column chromatography, yielding ∼10 mg/L Aaß-agarase. The purified recombinant Aaß-agarase exhibited optimal activity at pH 7 and 40°C, and its activity was strongly inhibited by Cu2+, Mn2+, Zn2+, and Al3+ ions. Furthermore, the KM and kcat values for purified Aaß-agarase were ∼0.02 mM and ∼45/s, respectively. These kinetic values were up to approximately 15-100-fold lower than the KM values reported for other agarases and approximately 7-30-fold higher than the kcat/KM values reported for other agarases, indicating that recombinant Aaß-agarase exhibited good substrate-binding ability and high catalytic efficiency. These results demonstrated that the E. coli expression system was capable of producing recombinant Aaß-agarase in an active form, at a high yield, and with attributes useful in the relevant industries.

Importance of Glu53 in the C-terminal region of brazzein, a sweet-tasting protein.

BACKGROUND: The sweetness of brazzein, one of the known sweet proteins, is dependent on charges and/or structures of its specific amino acid side chains. As the residues in the C-terminus of brazzein are known to play a critical role in sweetness, the currently unknown function of Glu53 requires further study. RESULTS: To identify important residues responsible for the sweetness of the protein brazzein, four mutants of the Glu53 residue in the C-terminal region of des-pE1M-brazzein, which lacks the N-terminal pyroglutamate, were constructed using site-directed mutagenesis. Mutations of Glu53 substitution to Ala or Asp significantly decreased the sweetness. On the other hand, a Lys mutation resulted in a molecule with sweetness similar to that of des-pE1M-brazzein. Mutation of Glu53 to Arg resulted in a molecule significantly sweeter than des-pE1M-brazzein, which agrees with previous findings showing that mutation with positively charged residues results in a sweeter protein. CONCLUSION: Our results suggest that the residue at position 53 is crucial for the sweetness of brazzein, which may be interacting with the sweet-taste receptor. © 2015 Society of Chemical Industry.

Guggulsterone and bexarotene induce secretion of exosome-associated breast cancer resistance protein and reduce doxorubicin resistance in MDA-MB-231 cells.

Many breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP/ABCG2). Here we show that incubation of human breast cancer MDA-MB-231 cells with farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, a sphingolipid known to induce exosome secretion. The gug+bex combination reduced cellular levels of BCRP to 20% of control cells by inducing its association and secretion with exosomes. Exogenous C6 ceramide also induced secretion of BCRP-associated exosomes, while siRNA-mediated knockdown or GW4869-mediated inhibition of neutral sphingomyelinase 2 (nSMase2), an enzyme generating ceramide, restored cellular BCRP. Immunocytochemistry showed that ceramide elevation and concurrent loss of cellular BCRP was prominent in Aldefluor-labeled breast cancer stem-like cells. These cells no longer excluded the BCRP substrate Hoechst 33342 and showed caspase activation and apoptosis induction. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death by more than fivefold. Taken together, our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy.

Antioxidant, anti-inflammatory, and anti-allergic activities of the sweet-tasting protein brazzein.

Sweet-tasting proteins may be useful as low-calorie sugar substitutes in foods, beverages, and medicines. Brazzein is an attractive sweetener because of its high sweetness, sugar-like taste, and good stability at high temperature and wide pH ranges. To investigate the bioactivities of brazzein, the antibacterial, antifungal, antioxidant, anti-inflammatory, and anti-allergic activities were determined in vitro. Brazzein showed no antibacterial and antifungal activities, although it showed approximately 45% or greater similarity to defensin, which has antimicrobial effects, and drosomycin, which is used as an antifungal agent. However, brazzein exhibited strong antioxidant effects, showing ABTS radical scavenging activity (IC50=12.55µM) and DPPH activity (IC50>30µM). Brazzein also showed anti-inflammatory activity and anti-allergic activity in a ß-hexosaminidase assay (IC50>15µM) and cyclooxygenase-2 inhibition assay (IC50=12.62µM), respectively. These results suggest that brazzein has antioxidant, anti-inflammatory, and anti-allergic activities and considerable potential as a functional sweetener.

Improved Secretory Production of the Sweet-Tasting Protein, Brazzein, in Kluyveromyces lactis.

Brazzein is an intensely sweet protein with high stability over a wide range of pH values and temperatures, due to its four disulfide bridges. Recombinant brazzein production through secretory expression in Kluyveromyces lactis is reported, but is inefficient due to incorrect disulfide formation, which is crucial for achieving the final protein structure and stability. Protein disulfide bond formation requires protein disulfide isomerase (PDI) and Ero1p. Here, we overexpressed KlPDI in K. lactis or treated the cells with dithiothreitol to overexpress KlERO1 and improve brazzein secretion. KlPDI and KlERO1 overexpression independently increased brazzein secretion in K. lactis by 1.7-2.2- and 1.3-1.6-fold, respectively. Simultaneous overexpression of KlPDI and KlERO1 accelerated des-pE1M-brazzein secretion by approximately 2.6-fold compared to the previous system. Moreover, intracellular misfolded/unfolded recombinant des-pE1M-brazzein was significantly decreased. In conclusion, increased KlPDI and KlERO1 expression favors brazzein secretion, suggesting that correct protein folding may be crucial to brazzein secretion in K. lactis.

The 5XFAD Mouse Model of Alzheimer's Disease Exhibits an Age-Dependent Increase in Anti-Ceramide IgG and Exogenous Administration of Ceramide Further Increases Anti-Ceramide Titers and Amyloid Plaque Burden.

We present evidence that 5XFAD Alzheimer's disease model mice develop an age-dependent increase in antibodies against ceramide, suggesting involvement of autoimmunity against ceramide in Alzheimer's disease pathology. To test this, we increased serum anti-ceramide IgG (2-fold) by ceramide administration and analyzed amyloid plaque formation in 5XFAD mice. There were no differences in soluble or total amyloid-ß levels. However, females receiving ceramide had increased plaque burden (number, area, and size) compared to controls. Ceramide-treated mice showed an increase of serum exosomes (up to 3-fold using Alix as marker), suggesting that systemic anti-ceramide IgG and exosome levels are correlated with enhanced plaque formation.

Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3.

Cilia are important organelles formed by cell membrane protrusions; however, little is known about their regulation by membrane lipids. We characterize a novel activation mechanism for glycogen synthase kinase-3 (GSK3) by the sphingolipids phytoceramide and ceramide that is critical for ciliogenesis in Chlamydomonas and murine ependymal cells, respectively. We show for the first time that Chlamydomonas expresses serine palmitoyl transferase (SPT), the first enzyme in (phyto)ceramide biosynthesis. Inhibition of SPT in Chlamydomonas by myriocin led to loss of flagella and reduced tubulin acetylation, which was prevented by supplementation with the precursor dihydrosphingosine. Immunocytochemistry showed that (phyto)ceramide was colocalized with phospho-Tyr-216-GSK3 (pYGSK3) at the base and tip of Chlamydomonas flagella and motile cilia in ependymal cells. The (phyto)ceramide distribution was consistent with that of a bifunctional ceramide analogue UV cross-linked and visualized by click-chemistry-mediated fluorescent labeling. Ceramide depletion, by myriocin or neutral sphingomyelinase deficiency (fro/fro mouse), led to GSK3 dephosphorylation and defective flagella and cilia. Motile cilia were rescued and pYGSK3 localization restored by incubation of fro/fro ependymal cells with exogenous C24:1 ceramide, which directly bound to pYGSK3. Our findings suggest that (phyto)ceramide-mediated translocation of pYGSK into flagella and cilia is an evolutionarily conserved mechanism fundamental to the regulation of ciliogenesis.

Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide.

We show here that human embryonic stem (ES) and induced pluripotent stem cell-derived neuroprogenitors (NPs) develop primary cilia. Ciliogenesis depends on the sphingolipid ceramide and its interaction with atypical PKC (aPKC), both of which distribute to the primary cilium and the apicolateral cell membrane in NP rosettes. Neural differentiation of human ES cells to NPs is concurrent with a threefold elevation of ceramide-in particular, saturated, long-chain C16:0 ceramide (N-palmitoyl sphingosine) and nonsaturated, very long chain C24:1 ceramide (N-nervonoyl sphingosine). Decreasing ceramide levels by inhibiting ceramide synthase or neutral sphingomyelinase 2 leads to translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora kinase A (AurA). Inhibition of aPKC, AurA, or a downstream target of AurA, HDAC6, restores ciliogenesis in ceramide-depleted cells. Of importance, addition of exogenous C24:1 ceramide reestablishes membrane association of aPKC, restores primary cilia, and accelerates neural process formation. Taken together, these results suggest that ceramide prevents activation of HDAC6 by cytosolic aPKC and AurA, which promotes acetylation of tubulin in primary cilia and, potentially, neural processes. This is the first report on the critical role of ceramide generated by nSMase2 in stem cell ciliogenesis and differentiation.

Characterization of human tyrosinase ectodomain expressed in Escherichia coli.

The human tyrosinase ectodomain has been expressed in Escherichia coli as a soluble form and purified by immobilized metal affinity column chromatography. The ectodomain exhibited tyrosinase activities toward the hydroxylation and oxidation reactions. Biochemical properties of the ectodomain appeared to be distinct from those of the human tyrosinase, although common features were retained.